1. Field of the Invention
This invention relates to thrombin inhibitors and substrates.
2. Description of the Related Art
Thrombin, the last enzyme in the coagulation system, cleaves soluble fibrinogen to fibrin, which is then crosslinked and forms an insoluble gel forming the matrix for a thrombus. When a vessel is damaged, the above process is necessary to stop bleeding. Under normal circumstances there is no measurable amount of thrombin present in plasma. Increase of the thrombin concentration can result in formation of clots, which can lead to thromboembolic disease, one of the most common serious medical problems of our time.
Thrombin contributes to haemostatic control by means of several biological reactions. In addition to its primary function, the conversion of fibrinogen to fibrin, thrombin activates Factor XIII, which is responsible for the crosslinking of fibrin. Thrombin also acts by means of a positive feedback mechanism involving the activation of Factors V and VIII, which both are necessary for its own formation from prothrombin. Thrombin has another essential role: its binding to platelets initiates platelet release and aggregation which is responsible for primary haemostasis.
Fibrinolysis is the process which causes an enzymatic dissolution of fibrinogen and fibrin clots. Plasma contains a protein, plasminogen, which under the influence of various activators is converted to plasmin, a proteolytic enzyme, the activity of which resembles that of fibrin. Plasmin breaks down fibrin to fibrin degradation products.
Under normal conditions, the fibrinolysis system is in balance with the coagulation system. Small thrombi formed in the blood stream can be dissolved enzymatically and the circulation through the vessels can be restored by the activation of the fibrinolytic system in the body. If the fibrinolytic activity is too high, it may cause or prolong bleeding and if it is too low compared to the activity of the coagulation system, there is a risk of thrombosis.
The reactions of thrombin are further controlled by natural inhibitors in plasma. The most important of these are antithrombin III and heparin. These two compounds have been isolated and are therapeutically and prophylactically used in conditions where there is an imbalance in the haemostatic mechanisms with risk for prothrombin activation.
Mainly two types of therapeutic agents are used for the prevention of thrombosis. The heparins act by accelerating the inhibition of thrombin by antithrombin III. Coumarin derivatives, the oral anticoagulants, e.g. Warfarin, prevent the generation of thrombin by blocking the post-translational vitamin K-dependent xcex3-carboxylation in the synthesis of prothrombin. Neither Heparin nor Warfarin are ideal. Heparin must be given parenterally and as it functions as a cofactor to antithrombin III it has no effect without this inhibitor. The effect of Warfarin develops very slowly and individual doses must be adjusted by frequent tests. None of these anticoagulants is specific for thrombin, they also inhibit other serine proteases and both of them may cause bleeding if the doses are not correctly balanced.
Thus, direct acting, specific thrombin inhibitors, having oral activity would be useful alternatives to the above anticoagulants. Much research in this area has resulted in the synthesis of different kinds of inhibitors of thrombin.
By imitating amino acid sequences of fibrinogen, the important natural substrate of thrombin, several good short peptide substrates for thrombin have been synthesized. The very first developed sequence with affinity for the active site of thrombin was Phexe2x80x94Valxe2x80x94Arg [1] which mimics the fibrinogensequence preceding the bond split by thrombin. This sequence has later been improved to give Dxe2x80x94Phexe2x80x94Proxe2x80x94Arg and Dxe2x80x94Phexe2x80x94Pipxe2x80x94Arg which have been used in chromogenic substrates, e.g. Dxe2x80x94Phexe2x80x94Proxe2x80x94Argxe2x80x94pNA and Dxe2x80x94Phexe2x80x94Pipxe2x80x94Argxe2x80x94pNA [1] and in inhibitors of thrombin, e.g. the peptide aldehyde Dxe2x80x94Phexe2x80x94Proxe2x80x94Argxe2x80x94H [2], the irreversible inhibitor Dxe2x80x94Phexe2x80x94Proxe2x80x94Argxe2x80x94CH2Cl [3], inhibitors with a ketomethylene bond e.g. Dxe2x80x94Phexe2x80x94Proxe2x80x94Argxe2x80x94kxe2x80x94Glyxe2x80x94piperidide [4] and in the recently synthesized peptide boronic acid inhibitors e.g. Zxe2x80x94Dxe2x80x94Phexe2x80x94Proxe2x80x94boroArg [5] and the nitrile: Bocxe2x80x94Dxe2x80x94Phexe2x80x94Proxe2x80x94ArgCN [6].
Thus, Dxe2x80x94Phexe2x80x94Proxe2x80x94Arg has been considered the best sequence for about 15 years, and it has been shown to have very good affinity for the active site of thrombin, in substrates (Km around 10xe2x88x926M) as well as in inhibitors (Ki 10xe2x88x927M to 10xe2x88x929M).
We have now found that by exchanging Phe, in the Dxe2x80x94Phexe2x80x94Proxe2x80x94Arg sequence, for some unnatural, aromatic amino acids, with a specified structure, and by using these new sequences to construct novel substrates and inhibitors we obtained significantly improved substrate and inhibitor properties. The new substrates show better kinetic constants (Km and kcat) and the inhibitors better inhibition constant (Ki).
Reduction of blood pressure is a side effect observed in many of the previous thrombin inhibitors containing Arg or Arg analogues like Gpa and Apa [7]. This side effect which in some compounds can be disturbingly serious is believed to depend on the positively charged guanidino or amidino group of the side chain of Arg or its analogues. Surprisingly, this side effect of inhibitors in the present application is markedly reduced even when the inhibitors have an Arg or Arg analogue.
We have also surprisingly found that by changing the side chain to a non-basic alkyl or alkylaryl group of a certain size, the affinity for thrombin is still very good although the affinity for other serine proteases is greatly reduced, i.e. these inhibitors/substrates are more specific for thrombin than corresponding compounds containing Arg. With this non-basic side chain the blood pressure lowering side effect is greatly reduced.
The present invention provides thrombin inhibitors and substrates derived from Dxe2x80x94Phexe2x80x94Proxe2x80x94Arg or its analogues wherein Phe is substituted by 
and Arg may be substituted by 
Suitably, the inhibitors/substrates are of formula I, in which 
X=H, CH3 or an N-protecting group, e.g. Ac, Bz, Cbz, Boc;
Y=[CH2]nxe2x80x94Q, 
where Q=H, amino, amidino, imidazole, guanidino or isothioureido and n=1-5, preferably 3-5, or C3-C9 alkyl and C5-C10 aryl or alkylaryl optionally substituted by up to three groups selected from hydroxy and C1-C4 alkoxy; 
R1=H, OH, CH2Cl, CH2xe2x80x94CH2xe2x80x94COxe2x80x94pip, CF2xe2x80x94CF2xe2x80x94COxe2x80x94pip, 
CH2xe2x80x94CH2xe2x80x94COxe2x80x94Proxe2x80x94NHEt, CF2xe2x80x94CF2xe2x80x94COxe2x80x94Proxe2x80x94NHEt or a chromophoric group e.g. pNA, MCA,
R2 and R3 may be the same or different and are selected from the group consisting of OH, OR6 and NR6R7, or R2 and R3 taken together represent the residue of a diol; where R6 and R7, which may be the same or different, are C1-C10 alkyl, phenyl or C6-C10 arylalkyl,
R4 and R5 may be the same or different and are selected from R2, R3, Glyxe2x80x94pip, Alaxe2x80x94pip or Glyxe2x80x94Proxe2x80x94NHEt; 
Ar1 and Ar2 may be the same or different and are selected from the group consisting of phenyl, thienyl, pyridyl, naphthyl, thionaphthyl, indolyl and saturated groups corresponding to these, optionally substituted by up to three groups selected from C1-C3 alkyl and C1-C3 alkoxy,
L1 and L2 may be the same or different and are selected from the group consisting of CH2, CH2xe2x80x94CH2, Oxe2x80x94CH2, Sxe2x80x94CH2,
Arxe2x80x94L taken together may mean H, diphenyl-methyl, fluorenyl or saturated groups corresponding to these, but one of the Arxe2x80x94L cannot be H when the other Arxe2x80x94L means H or benzyl; 
or its C1-C3 alkyl substituted derivatives, where R8xe2x95x90CH2, CH2xe2x80x94CH2, Sxe2x80x94CH2, Sxe2x80x94C(CH3)2 or CH2xe2x80x94CH2xe2x80x94CH2.
Preferably the Phe substitute is Dpa, Nal or Dba and preferable Arg substitute includes Irg, Gpa, Apa and non-basic amino acids such as Pgl, Mbg, Chg.
Examples of compounds which may be preferably used in the invention include:
Acxe2x80x94Dxe2x80x94xcex2Nalxe2x80x94Proxe2x80x94boroArg pinanediol ester
Zxe2x80x94Dxe2x80x94Dpaxe2x80x94Proxe2x80x94boroIrg pinanediol ester
Zxe2x80x94Dxe2x80x94Dpaxe2x80x94Proxe2x80x94boroPgl pinacol ester
Acxe2x80x94Dxe2x80x94xcex2Nalxe2x80x94Proxe2x80x94boroMbg pinanediol ester
CH3xe2x80x94Dxe2x80x94Dpaxe2x80x94Proxe2x80x94Argxe2x80x94H
Bocxe2x80x94Dxe2x80x94Dpaxe2x80x94Proxe2x80x94Gpaxe2x80x94H
CH3xe2x80x94Dxe2x80x94Dpaxe2x80x94Thixe2x80x94Mbgxe2x80x94H
Hxe2x80x94Dxe2x80x94Dpaxe2x80x94Proxe2x80x94Argxe2x80x94kxe2x80x94Glyxe2x80x94pip
Zxe2x80x94Dxe2x80x94Dpaxe2x80x94Proxe2x80x94Argxe2x80x94CH2Cl
Bocxe2x80x94Dxe2x80x94Dpaxe2x80x94Proxe2x80x94ArgCN
Hxe2x80x94Dpaxe2x80x94Proxe2x80x94ArgP (OPh)2 
Hxe2x80x94Dxe2x80x94xcex2Nalxe2x80x94Proxe2x80x94PglP (OPh)xe2x80x94Glyxe2x80x94pip
Hxe2x80x94Dxe2x80x94Dpaxe2x80x94Pipxe2x80x94Argxe2x80x94pNA
Hxe2x80x94Dxe2x80x94xcex2Nalxe2x80x94Proxe2x80x94Chgxe2x80x94pNA
Further examples of compounds which may be preferably used in the invention are those listed in Examples 10 to 22 below.
Inhibition data for some of the new compounds are shown in Tables 1-7. The advantages of replacing Phe by amino acids according to the invention are clearly shown in the Ki values, which are generally 3 to 10 times better for the new compounds, as well as in the prolongation of the thrombin time. The importance of the D-form of the N-terminal amino acid is also evident from Table 1. The drastic reduction of the blood pressure lowering side-effect with compounds according to the invention is shown in Table 2.
Those compounds of the invention which are thrombin inhibitors have anti-thrombogenic properties and may be employed for indications when an anti-thrombogenic agent is indicated. Generally, these compounds may be administered orally or parenterally to a host to obtain an anti-thrombogenic effect. In the case of larger mammals such as humans, the compounds may be administered alone or in combination with pharmaceutical carrier or diluent at a dose of from 0.02 to 15 mg/Kg of body weight and preferably 1-10 mg/Kg to obtain the anti-thrombogenic effect, and may be given as single dose or in divided doses or as a sustained release formulation. When an extracorporeal blood loop is to be established for a patient, 0.1-1 mg/Kg may be administered intravenously. For use with whole blood from 1-10 mg per liter may be provided to prevent coagulation. Pharmaceutical diluents are well known and include sugars, starches and water which may be used to make tablets, capsules, injectable solutions and the like. The compounds of the invention may be added to blood for the purpose of preventing coagulation of the blood in blood collecting or distribution containers, tubing or implantable apparatus which comes in contact with blood.
The advantages of the compounds of the invention include oral activity, rapid onset of activity and low toxicity. In addition, these compounds may have special utility in the treatment of individuals who are hypersensitive to compounds such as heparin.
In the following examples, the symbols have the following meanings:
The following non-limiting examples illustrate the preparation of the compounds in this invention.
The synthesis of some of the different inhibitor types are outlines in Schemes 1 to 8 and the detailed descriptions are given in the examples below.
HPLC
The following conditions were adopted for the analysis of most of the synthetic compounds on reversed-phase HPLC (RP-HPLC): column; SuperPac Pep-S (4xc3x97250 mm), eluant; A=water containing 0.1% TFA, B=acetonitrile containing 0.1% TFA, gradient; 50% to 90% B in A in 25 min, flow rate; 1.0 ml/min, detection; UV absorbance at 210 nm.
TLC
Thin layer chromatography (TLC) was carried out on the following compounds using precoated silica plates (Merck, F254) in the following systems: A, Chloroform-ethyl acetate (2:1); B, chloroform-methanol-acetic acid (20:4:1); C, n-butanol-acetic acid-ethyl acetate-water (1:1:1:1); D, chloroform-methanol (9:1); E, pyridin-ethyl acetate-acetic acid-water (5:5:1:3); F, chloroform-methanol-ammonia (1M) (60:35:5). The spots were visualized by ninhydrin and chlorine-dicarboxidine spray reagents (C. M. Swahn and J. Gyllander, J. Chromatogr. (1979) 170, 292:
NMR spectra
Magnetic resonance spectra were recorded at 250 MHz using a Bruker instrument.